Long noncoding RNA Mhrt alleviates angiotensin II-induced cardiac hypertrophy phenotypes by mediating the miR-765/Wnt family member 7B pathway

Abstract Long noncoding RNAs (lncRNAs) are known to participate in the pathological process of cardiac hypertrophy. This study aimed to investigate the function of the lncRNA, myosin heavy-chain associated RNA transcript (Mhrt), in cardiac hypertrophy and its possible mechanism of action. Adult mouse cardiomyocytes were treated with angiotensin II (Ang II) and transfected with Mhrt; cardiac hypertrophy was evaluated by estimating atrial natriuretic peptide, brain natriuretic peptide, and beta-myosin heavy-chain levels, and cell surface area by reverse transcription-quantitative polymerase chain reaction, western blotting, and immunofluorescence staining. The interaction between the Mhrt/Wnt family member 7B (WNT7B) and miR-765 was assessed using a luciferase reporter assay. Rescue experiments were performed by analyzing the role of the miR-765/WNT7B pathway underlying the function of Mhrt. The results indicated that Ang II induced hypertrophy of cardiomyocytes; however, overexpression of Mhrt alleviated the Ang II-induced cardiac hypertrophy. Mhrt acted as a sponge for miR-765 to regulate the expression of WNT7B. Rescue experiments revealed that the inhibitory effect of Mhrt on myocardial hypertrophy was abolished by miR-765. Additionally, the knockdown of WNT7B reversed the suppression of myocardial hypertrophy induced by downregulating miR-765. Taken together, Mhrt alleviated cardiac hypertrophy by targeting the miR-765/WNT7B axis.


Introduction
In an adult heart, cardiac hypertrophy is characterized by increased sizes of individual cardiomyocytes, which reduces ventricular wall pressure and maintains the normal function and efficiency of the heart [1,2]. In addition to physiological hypertrophy, pathological cardiac hypertrophy is associated with cardiac dysfunction, increased interstitial fibrosis, and cell death, which usually involves heart failure and sudden death [3,4]. As the density of capillaries decreases, blood supply is insufficient, leading to the transformation of physiological cardiac hypertrophy to pathological hypertrophy [5]. In recent years, many mediators involved in the processes of cardiac hypertrophy have been reported, such as the G protein, renin-angiotensin system, and the PI3K/AKT, MAPK, and NF-kappaB pathways [6]; however, the regulatory mechanisms remain unclear. As gene therapy is a novel therapeutic approach [7], it is necessary to explore more effective targets for the treatment of cardiac hypertrophy.
Long noncoding RNAs (lncRNAs) are noncoding RNA transcripts that are longer than 200 nucleotides. Increasing evidence suggests that dysregulation of lncRNAs is linked to cardiac hypertrophy, especially physiological hypertrophy [8,9]. In the pathological process, aberrant expression of lncRNAs is associated with sarcomere formation, calcium processing, and mitochondrial dysfunction, leading to genetic mutations, stress overload, inflammation, and endocrine abnormalities [10]. Additionally, lncRNAs are associated with cardiac remodeling [11]. Myosin heavy-chain associated RNA transcript (Mhrt), also named Myheart, is a transcript located on the Myh7 gene. Mhrt is cardiac-specific, found only in cardiomyocytes, and is present at low levels in fetal hearts but is abundantly present in adult hearts [12]. The level of Mhrt is elevated in cardiac myocytes of hearts that have undergone acute myocardial infarction and protects cardiomyocytes from apoptosis [13]. Low expression of Mhrt indicates a poor survival rate in patients with chronic heart failure [14]. In cardiac hypertrophy, Mhrt can inhibit myocardin expression and attenuate disease progression [15]. However, the regulatory roles of Mhrt in cardiac hypertrophy remain unclear, and further research is needed.
In this study, we aimed to explore the functions of Mhrt in the regulatory mechanisms underlying cardiac hypertrophy. We hypothesized that overexpression of Mhrt attenuated angiotensin II (Ang II)-induced cardiac hypertrophy. Additionally, Mhrt could bind to the microRNA miR-765 to regulate the expression of the Wnt family member 7B (WNT7B). Furthermore, the function of the downstream regulatory pathway miR-765/WNT7B was explored. The goal of the study provided a potential option, mediating the Mhrt/miR-765/WNT7B axis, to treat pathological cardiac hypertrophy.

Bioinformatics analysis
The starbase online database (https://starbase.sysu.edu. cn/) was used to select the target miRNAs of Mhrt and the binding sites between miR-765 and the 3′-UTR of Mhrt. The TargetScan (https://www.targetscan.org/vert_80/) and miRDB (https://mirdb.org/) databases were used to obtain the genes targeted by miR-756 and the binding sites between miR-765 and the WNT7B. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were used to identify the significant pathways of target genes.

Animal model
The animal experimental protocol was approved by the Ethics Committee of the Strategic Support Force Medical Center. Male C57BL/6 mice (6-8 weeks old, 20-22 g) were divided into control and Ang II groups (six mice per group). The mice in the Ang II group were administered 1.46 mg/kg/day Ang II (Sigma-Aldrich, St. Louis, MO, USA) for 2 weeks by implanting osmotic mini-pumps in the peritoneal cavity. Similarly, mice in the control group were administered saline for 2 weeks. All the mice were sacrificed by cervical dislocation, and their hearts were removed and weighed after washing with PBS.

Hematoxylin-eosin (H&E) staining assay
H&E staining was performed as described previously [16]. Isolated hearts were fixed with 4% paraformaldehyde for 24 h. Then, the cardiac tissues were cut into 5 μm sections after paraffin embedding. All sections were stained with hematoxylin for 10 min and eosin for 1 min. The stained sections were visualized under a microscope.

Western blotting
All the kits or reagents for western blotting were obtained from Elabscience (Wuhan, China). Cells were lysed using the RIPA lysis buffer, and the protein concentration was estimated using the BCA Protein Concentration Detection ) was employed to separate proteins, which were then transferred onto PVDF membranes. The membranes were blocked for 1.5 h in 5% skim milk. The membranes were then incubated with primary antibodies (anti-ANP: ab209232, 1:1,000; anti-BNP: ab236101, 1:2,000; anti-β-MHC: ab180779, 1:2,000; and anti-β-actin: ab8227, 1:5,000; Abcam, Cambridge, CA, USA) overnight at 4°C and then incubated with secondary antibodies (Catalog No. E-AB-1003; Elabscience) for 1 h at 25°C. Protein bands were visualized using an ECL luminous detection solution (Catalog No. E-BC-R347). Gray analysis was performed by normalizing to GAPDH levels.

Immunofluorescence (IF) staining
IF staining was performed as described previously [16].
After 48 h of transfection, the cells were fixed with 4% formaldehyde and permeabilized in 0.5% Triton X-100 for 20 min at room temperature. The cells were then washed with PBS and blocked with normal goat serum for 30 min. Primary antibody (α-actinin: ab90421, 1:100; Abcam) was added to the cells, and the cells were incubated overnight at 4°C. The next day, the cells were incubated with a secondary antibody (Goat Anti-Mouse IgG H&L [Alexa Fluor ® 594]: ab150116, 1:500; Abcam) at room temperature for 1 h. IF was detected using a fluorescence microscope (Olympus, Tokyo, Japan) after the cells were incubated with DAPI for 10 min. One hundred cells from three wells were randomly selected to quantify the surface area using Image-Pro Plus 6.0.

Luciferase reporter assay
The binding sites between Mhrt and miR-765 were predicted using the Starbase database, and the binding sites between miR-765 and WNT7B were predicted using the TargetScan database. HEK293T cells were seeded into 24-well plates. Mhrt-wild type (WT) and WNT7B-WT containing the predicted binding sites of miR-765 were inserted into pGL3 vectors, and their corresponding mutant (MUT) sequences were also cloned into pGL3 vectors. These recombinant plasmids were co-transfected with the miR-765 mimic and mimic NC into HEK293T cells using Lipofectamine 2000 Transfection Reagent (Invitrogen). After 48 h, relative luciferase activity was analyzed using the Dual-Luciferase Reporter Assay System (Catalog No. E1960; Promega, Madison, WI, USA).

Statistical analysis
All experiments were repeated at least three times, and the data are represented as mean ± SD. GraphPad Prism 6.0 software (GraphPad, CA, USA) was used for the analysis. Significant differences were determined using the Student's t-test (between two groups) and one-way ANOVA (between multiple groups). P < 0.05 was considered to be statistically significant.
The results of RT-qPCR showed that the mRNA expression levels of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and beta-myosin heavy chain (β-MHC) in the Ang II-treated group were significantly increased than those in the control group (Figure 1a). Similarly, the protein levels of ANP, BNP, and β-MHC were also significantly higher in Ang II-treated cells (Figure 1b). The most significant Ang II concentration (10 −7 mol/l) was used in subsequent cell experiments. The results of the H&E staining assay showed that the size of the cardiomyocyte was increased (Figure 1c). In addition, Ang II treatment caused the heart to grow bigger, which in turn increased the ratio of heart weight/body weight ( Figure 1d). Moreover, the expression of Mhrt was reduced in cardiomyocytes treated with Ang II, as analyzed by RT-qPCR (Figure 1e).

Overexpressed Mhrt alleviated Ang IIinduced cardiac hypertrophy
To explore the functions of Mhrt, cardiac myocytes were transfected with the Mhrt vector and treated with Ang II. The RT-qPCR data indicated that Mhrt was significantly upregulated after transfection of the Mhrt overexpression vector (Figure 2a). Meanwhile, ANP, BNP, and β-MHC were elevated by Ang II both at the mRNA and protein levels, which were rescued by overexpressed Mhrt (Figure 2b and c). Ang II remarkably increased the cell surface area, which was significantly alleviated by overexpressed Mhrt (Figure 2d).

miR-765 functioned as the target of Mhrt
Through the starbase database (https://starbase.sysu. edu.cn/), we obtained the top-10 target miRNAs of  Mhrt, and after Mhrt overexpression, just the miR-765 expressions were decreased while other miRNAs showed no difference (Figure 3a). Besides, the binding sites between miR-765 and the 3′-UTR of Mhrt were obtained from the starbase database, and Mhrt-MUT sequences were designed (Figure 3b).In addition, the transfection efficiency of si-Mhrt was tested, and we found the Mhrt levels were significantly decreased after si-Mhrt transfection. Si-Mhrt 3# was selected for the next experiments because of the best transfection efficiency (Figure 3c). Luciferase reporter assay demonstrated that co-transfection with the miR-765 mimic and Mhrt 3′-UTR WT significantly reduced luciferase activity, while in the Mhrt-MUT group, a significant change in the luciferase activity was not observed (Figure 3d). Silencing Mhrt (si-Mhrt 2# and si-Mhrt 3# transfection) significantly increased the expression of miR-765 but the overexpression of Mhrt remarkably reduced miR-765 expression (Figure 3e). In Ang II-treated cells, the miR-765 expression was significantly higher than that in the control group (Figure 3f).

Inhibitory effect of Mhrt on cardiac hypertrophy was abolished by miR-765 in cardiomyocytes
In transfected cells, Ang II increased the miR-765 level, which was suppressed by Mhrt; however, this suppression was abrogated by miR-765 mimic (Figure 4a). Furthermore, overexpression of miR-765 partially abolished the inhibition of ANP, BNP, and β-MHC by Mhrt in Ang IIinduced hypertrophic cells (Figure 4b and c). The cardiomyocyte phenotype was altered by Mhrt, which was rescued by the miR-765 mimic in Ang II-induced myocardial hypertrophy (Figure 4d).

Knockdown of WNT7B reversed the effects of miR-765 on cardiac hypertrophy
After transfection, WNT7B expression was decreased by Ang II but was increased by miR-765 inhibitor; this increase was subsequently abolished by si-WNT7B ( Figure 6a). The levels of ANP, BNP, and β-MHC were repressed by the downregulation of miR-765 in Ang IIinduced hypertrophic cells; this effect was abrogated by silencing WNT7B (Figure 6b and c). In addition, the inhibitory effect on the cardiomyocyte size induced by the miR-765 inhibitor was reversed by si-WNT7B (Figure 6d). Additionally, we also performed these experiments using another si-WNT7B 2# and obtained the same results ( Figure A1). Moreover, we found that WNT7B knockdown, like ANG treatment, also induced hypertrophy of cardiomyocytes ( Figure A2).

Discussion
Increasing evidence has demonstrated that lncRNAs are associated with cardiovascular diseases, including cardiac hypertrophy [8,9]. In the present study, we explored the function of Mhrt in cardiac hypertrophy and found that Mhrt could alleviate Ang II-induced cardiac hypertrophy via the miR-765/WNT7B pathway. The pathological cardiac hypertrophy process is usually accompanied by the release of ANP, BNP, and β-MHC, as well as enlargement of the surface area of cardiomyocytes [17,18]. In this study, we found that Ang II enhanced ANP, BNP, and β-MHC, and also increased the cell surface area, suggesting that Ang II induced hypertrophy of cardiomyocytes.
Dysregulation of Mhrt is associated with the pathogenesis of cardiomyopathy, such as cardiac hypertrophy, heart failure, and cardiac fibrosis [19,20]. Mhrt is highly enriched in the nucleus of cardiomyocytes and downregulated by pressure overload. Inhibition of Mhrt transcription and restoring Mhrt levels to pre-stress levels protect the heart from hypertrophy and failure [12]. Previous studies have shown that different mechanisms of Mhrt are involved in cardiac hypertrophy. For example, maintaining Mhrt at pre-stress levels protects the heart from hypertrophy by inhibiting the activation of the Brg1-Hdac-Parp chromatin repressor complex [12]. Besides, an interaction between Mhrt and myocardin could suppress cardiac hypertrophy, including the effect of Mhrt on the acetylation of myocardin and myocardin-activating Mhrt transcription [21]. Unlike the findings mentioned above, Xu et al. [15] suggested that Mhrt represses myocardin expression by regulating the miR-145a-5p/KLF4 pathway, leading to myocardial hypertrophy. Additionally, several lncRNAs also regulate cardiac hypertrophy, such as lncRNA AK006774 and NEAT1 [22,23]. Based on these previous studies, we investigated the function of Mhrt in cardiac hypertrophy by estimating ANP, BNP, and β-MHC levels, and the cell surface area. We found that Mhrt levels decreased in Ang II-induced cardiac hypertrophy. Overexpressed Mhrt inhibited ANP, BNP, and β-MHC levels, as well as cell surface area in Ang II-treated myocardial cells, suggesting that Mhrt could alleviate Ang II-induced cardiac hypertrophy.
MiR-765 is a microRNA located on chromosome 1. Many previous studies have examined the role of miR-765 in numerous diseases. For instance, in malignancy, miR-765 commonly functions as a tumor suppressor or tumor promoter and acts as a prognosis biomarker [24][25][26]. miR-765 inhibited the protective effect of cerebral ischemia/reperfusion injury induced by the knockdown of lncRNA FOXD3-AS1 [27]. Additionally, miR-765 is involved in cardiovascular diseases; it is found to be highly expressed in patients with coronary heart disease [28]. In geriatric coronary artery disease, miR-765 levels are observed to be upregulated; these values are important for the clinical diagnosis of this disease [29]. Moreover, high expression of miR-765 is conducive to heart failure [30]. In the present study, miR-765 was upregulated in Ang II-treated myocardial cells; however, Mhrt was able to sponge miR-765. Functionally, miR-765 abolishes the inhibitory effect of Mhrt on ANP, BNP, and β-MHC levels and induces enlargement of the cardiomyocyte surface area, suggesting that Mhrt attenuated Ang II-induced cardiac hypertrophy by sponging miR-765 in this study.
Wnt signaling is usually activated during cardiac development, cardiac hypertrophy, myocardial infarction, and heart failure [31]. WNT7B, a member of the Wnt family, encodes a Wnt signaling ligand. WNT7B has been reported to promote choroidal neovascularization, to be involved in vascular calcification, and is associated with pulmonary arteriolar remodeling induced by hypoxia [32][33][34]. However, the role of WNT7B in cardiac hypertrophy remains unknown. In the current study, WNT7B was identified to be a target of miR-765, and its level was downregulated in Ang II-induced cardiac hypertrophy. Furthermore, the knockdown of WNT7B abrogated the inhibitory effect on ANP, BNP, and β-MHC levels and cell surface area induced by the downregulation of miR-765. Taken together, the downregulation of miR-765 targeted WNT7B to suppress Ang II-induced cardiac hypertrophy.

Conclusion
LncRNA Mhrt is downregulated in cardiac hypertrophy. Overexpression of Mhrt protected against Ang II-induced cardiac hypertrophy via the miR-765/WNT7B axis. These findings suggest that mediation of the Mhrt/miR-765/ WNT7B axis can be used as a treatment strategy for pathological cardiac hypertrophy.
Funding information: This study was supported by the Capital Characteristic Clinic Project (No. Z181100001718015).

Conflict of interest:
The authors declare that they have no conflict of interest.
Data availability statement: The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Figure A2: WNT7B knockdown induced hypertrophy of cardiomyocytes. (a) Transfection efficiency of si-WNT7B was analyzed using RT-qPCR. (b) ANP, BNP and β-MHC mRNA expression levels were evaluated using RT-qPCR. (c) ANP, BNP, and β-MHC levels were evaluated using western blotting and quantified with GAPDH as an internal control. (d) Cell surface area was visualized using IF staining and quantified. **P < 0.01, ***P < 0.001.